A subset of patients with thyroid cancer has neck recurrences and metastases, is refractory to current treatments, and dies of the disease. BRAFV600E, the most frequent genetic alteration in both papillary (PTC) and anaplastic thyroid cancers (ATC), is implicated in progression from PTC to ATC. Our Gene Set Enrichment Analysis revealed that 7 of 17 gene sets up-regulated in BRAFV600E PTC were enriched in pro-metastatic extracellular matrix (ECM) proteins (e.g. thrombospondin-1 (TSP-1)) and receptors (integrins), including the integrin-linked kinases (i.e. focal adhesion kinase (FAK)) and the transcription factor (TF) HMGB1, which has been implicated in melanoma progression and metastasis. Knockdown of either TSP-1 or BRAFV600E inhibits cell proliferation, adhesion, migration/invasion, and metastasis in BRAFV600E-positive PTC and ATC cells and knockdown of TSP-1 significantly decreases levels of phospho(p)-ERK1/2, pFAK, and integrins. The novel selective BRAFV600E inhibitor PLX4720 shrinks tumor size in an orthotopic mouse model of ATC. However, persistence of the residual tumor and resumption of TSP-1, pERK1/2, and pFAK protein expression after 3 weeks of treatment suggest that metastatic thyroid cancer cells acquire resistance to BRAFV600E inhibition by up-regulating these proteins. Our objective is to identify BRAFV600E-dependent and -independent biomarkers for aggressive PTC by determining the essential signaling networks triggered by metastatic ECM proteins in BRAFV600E PTC cells. Specific Aim 1: To identify and assess potential biomarkers for PLX4720-resistant PTC by defining the molecular cascades by which TSP-1 stimulates ERK1/2 and FAK phosphorylation in human BRAFV600E PTC following inhibition of BRAFV600E. Preliminary data suggest that TSP-1 is associated with neck recurrence in BRAFV600E PTC. We will determine whether TSP-1 is a valid prognostic biomarker for PTC aggressiveness and establish an immunohistochemistry-based screening process suitable for clinical trials. To identify a larger panel of potential new prognostic biomarkers in BRAFV600E positive PTC, we plan to identify TSP-1 interactors. We will assess the correlation of some of these with TSP-1 expression and with clinico-pathological features of BRAFV600E positive PTC. To obtain a better understanding of BRAFV600E function in PTC progression, we will apply an unbiased proteomic analysis combined with functional assays to determine ECM protein interactions and intracellular signaling cascades in BRAFV600E PTC cells. Specific Aim 2: To explore whether HMGB1 is a biomarker for PTC aggressiveness. We will correlate HMGB1 expression with TSP-1 expression and clinico-pathological features of BRAFV600E PTC and explore whether HMGB1 regulates expression of TSP-1 or of TFs crucial for TSP-1 expression. The results of this study are likely to (i) identify new prognostic biomarkers of aggressive BRAFV600E positive PTC that can be assayed in biological fluids and PTC tissues to help monitor patients undergoing targeted therapies and enable earlier diagnosis of these thyroid cancers, and (ii) foster development of innovative therapies for PTC refractory to current treatments.